Friedreich ataxia (FRDA) is an autosomal recessive neuro- and cardio-degenerative disorder for which there are currently no established effective treatments. The identification of the disease gene in 1996 and the subsequent elucidation of the function of the encoded protein, frataxin, have opened the door to possible therapeutic approaches, including conventional high-throughput drug screening. This proposal describes a novel, complementary approach to the development of therapeutics for FRDA. We devised a method to construct a library that encodes random, short-hairpin-loop RNAs (shRNAs). This library can be used to identify shRNA sequences of therapeutic, therapeutic-targeting, and/or biological interest. The identification is based on functional selection, with effective sequences retrieved by PCR from cells that survive a particular condition or exhibit a predetermined phenotype. The design allows for hit-optimization of effective sequences, with re-selection for sequences with improved effects. Using primary FRDA fibroblasts, we developed screening and selection assays based on the critical role of mitochondrial dysfunction in the signs and symptoms of FRDA and on the sensitivity of FRDA cells to oxidative stress. With our live/dead selection assays, we can use our random shRNA-encoding library to identify shRNA sequences of benefit to FRDA cells. With our screening assays of mitochondrial function, we can confirm and prioritize these sequences. The primary objective of this proposal is to identify potential shRNA therapeutics. A secondary objective is to begin to understand the mechanisms of action of the shRNAs we identify. The Specific Aims are: 1. To identify shRNA sequences that allow survival under conditions lethal to primary FRDA fibroblasts but non-lethal to normal control cells. 2. To optimize the shRNA sequences identified in Aim 1. 3. To confirm and prioritize optimized shRNAs and begin to understand mechanisms of action. This proposal describes an approach to develop novel therapeutics for Friedreich ataxia using a random shRNA library. This approach has potential implications for the development of therapeutics for other genetic diseases, as well as for infectious diseases, and is therefore highly relevant to public health.